Mass-spectrometric resistance determination by growth measurement

ABSTRACT

The invention relates to a mass-spectrometric method to determine microbial resistances to antibiotics, in which the microbes are cultured in a medium comprising an antibiotic, and a mass spectrum of the microbes is acquired after they have been cultured. The method is characterized by the fact that any microbial growth taking place during the culture is mass-spectrometrically determined with the aid of a reference substance, which is added in a dosed amount and is co-measured in the mass spectrum, wherein a growth in microbes indicates the resistance to the antibiotic.

TECHNICAL FIELD

The invention relates to a mass-spectrometric method to determinemicrobial resistances to antibiotics.

DEFINITIONS

Instead of the statutory “unified atomic mass unit” (u), this documentuses the unit “dalton” (Da), which was added in the last (eighth)edition 2006 of the document “The International System of Units (SI)” ofthe “Bureau International des Poids et Mesures” on an equal footing withthe atomic mass unit; as is noted there, this was primarily done inorder to be able to use the units kilodalton, millidalton and similar.

For reasons of simplicity, only the term “proteins” is used in thisdocument, although in the preferred mass range of 3,000 to 15,000daltons it would often be better to call the proteins “peptides”. Thetransition from the lighter peptides to the heavier proteins is fluidand not unequivocally defined.

The term “microbes” is often used here as a short form for“microorganisms”. The singular “microbe” also means, as is usual ingeneral parlance, the microbial species as well as the individualmicrobial cell. The plural “microbes” means the microbial cells underanalysis.

BACKGROUND ART

Many species of microorganisms, particularly bacteria and unicellularfungi such as yeasts, can be identified nowadays quickly and with lowerror rates by means of mass spectrometry. The identification isroutinely done by computing similarity values between a mass spectrum ofthe disrupted (lysed) microbes, particularly their soluble proteins, andsimilar types of reference mass spectra of known microorganisms. If thesimilarity values exceed certain limit values, family, genus, speciesand even strain can be identified. This very fast and low-cost method ofidentifying microorganisms has proven to be extraordinarily successful,both in large scale studies and in the daily routine in manymicrobiological laboratories. Depending on the instrument, 48 to 384microbial samples can be determined at the same time; identificationtakes only minutes from the end of culturing a colony to theidentification. The method has very low error rates, much lower than theerror rates of conventional microbiological identification methods, andlower even than those of DNA analyses. There are meanwhile massspectrometers, associated evaluation programs and libraries of referencespectra on the market which are certified as IVD products for medicaldiagnostics in accordance with the German Medical Devices Act (MPG) andother, national or international regulations and guidelines.

As is usual in general parlance, the term “antibiotic” means apharmacologically active substance for the treatment of microbialinfectious diseases and also substances for disinfection. The successesof penicillin led to the search for and discovery of many otherantibiotics. There are broad-spectrum antibiotics, which are effectiveagainst many families of microbes, and narrow-spectrum antibiotics,which are specifically effective against individual microbe species.

Ever since penicillin was used as the first pharmacologically activesubstance, microbial strains have increasingly developed various typesof resistance to different types of antibiotics, or acquired them fromother microbes, i.e. they have acquired characteristics which allow themto weaken the effect of antibiotic substances or to neutralize theireffect completely. Resistances are unfortunately common meanwhile;microbes occurring in hospitals are today resistant in the main. In somecases, it is possible to predict the resistance of a microbe transmittedwithin the hospital to the antibiotics usually used in the hospital;this does not apply to infections which were acquired outside thehospital.

The success of a therapy for microbial infections, which are usuallylife-threatening in acute situations such as sepsis, or as a secondaryinfection during an existing primary illness (or primary infection),often depends on the first administration of an antibiotic beingeffective. Targeted administration requires not only that the pathogenis identified as quickly and correctly as possible, but also that itsresistance to different antibiotics is determined as quickly aspossible. The conventional determination of resistance consists of aculture in the presence of an antibiotic, but this unfortunately takes avery long time: 24 to 48 hours.

In addition to culturing in the presence of antibiotics, there are alsogenetic methods of determining resistances. There, a resistance isdetected by detecting known resistance genes in the genome of thepathogen in question. An advantage of the genetic methods consists inthe fact that the resistance genes can be amplified by techniques suchas polymerase chain reaction (PCR), and thus the time needed for theanalysis is no longer determined by the growth rate of the bacteria. Thedisadvantages are that they are more expensive than routine methods andare not functional tests. A resistance gene may be present, but not beexpressed, which means the bacterial strain under investigation is notresistant, but the method detects it as being resistant.

The patent DE 10 2006 021 493 B4 (V. M. Govorun and J. Franzen, 2006,corresponding to GB 2 438 066 B and U.S. Pat. No. 8,293,496 B2; called“Govorun” in the following) discloses mass-spectrometric methods for theresistance determination of microbes.

In one embodiment, protein profiles of the microbes aremass-spectrometrically measured and compared after being cultured inmedia with and without added antibiotics, for example. Here the microbesare cultured in centrifuge tubes with and without antibiotics, forexample. After culturing, the microbes are centrifuged out, rinsed, andthen disrupted with acids and acetonitrile in the centrifuge tubes sothat their soluble proteins are released. A small amount (around onemicroliter) of this liquid with disrupted microbial cells is preparedonto the sample support, dried and then coated with a small amount ofmatrix solution (also around one microliter). The dissolved proteins areembedded into the matrix crystals which are produced in the dryingprocess. The samples with matrix crystals and embedded proteins arebombarded with laser light pulses in the mass spectrometer, causing ionsof the protein molecules to be formed in the vaporization plasma.Measuring their time-of-flight in a time-of-flight mass spectrometerproduces the mass spectrum of the microbe, which essentially consists ofthe protein peaks.

The similarity between the two mass spectra allows only limitedconclusions to be drawn about the resistance of the microbes. Ifsusceptible microbes are only inhibited in their growth or killed,without being destroyed, as for example klebsiellae from the family ofthe enterobacteriaceae, the resulting mass spectrum is practicallyidentical to that of the microbes from media without antibiotics. Thisis because the formation of the mass spectra which are obtained usingionization by matrix-assisted laser desorption (MALDI) is only slightlydependent on the quantity of microbes in the sample. Sample preparationswith ten thousand microbes provide practically the same mass spectra assample preparations with ten million microbes, which is ideal for anidentification, but not for identifying the resistance. If merely thegrowth of the microbes is stopped, the same mass spectra result, becausethe only difference is in quantity, which does not show up in the massspectra.

As is explained further in the Govorun patent, the resistance can alsobe identified by adding a second type of microbe to the microbes underinvestigation; this second type provides a very different mass spectrum,is resistant, and definitely continues to grow in the presence of theantibiotic. The mass spectrum with the superimposed proteins of bothmicrobes should then show the differences in growth. Unfortunately, thismethod has proved to be not very practicable in routine work for variousreasons; it assumes at least rough quantitative determinations of themicrobes used, approximately equal growth rates in the culture mediumused, and that the reference microbes are resistant to many antibiotics.

The microbes whose resistance is to be determined are preferably presentin sufficient quantities in a sufficiently pure form. They can formcolonies on an agar, or also exist as microbes from a blood culture, forexample. With agar cultures, it is common practice to use microbes fromnot just one colony for the test, but to subject the microbes from atleast five colonies together to this test in order to identify thepossible presence of a resistant microbe among non-resistant microbes ofthe same species. A trained and experienced laboratory technician isgenerally able to recognize colonies of the same species of microbe andto harvest them. They must then be mixed and divided up for thecultures. Blood cultures naturally contain a mixture of the differentmicrobes which were transmitted in the infection.

As a rule, a resistance determination (including one according toGovorun) is often preceded by the identification of the microbes whichhave grown on the agar culture or in the blood culture. It is helpfulhere, especially for the method according to Govorun, to know themicrobe species and its growth rate. It is also usually the case thatthe antibiotics against which this microbe can be resistant are alsoknown. Furthermore, the minimum inhibitory concentrations (MIC) forsusceptible microbes are also usually known. This means that cultureswith these antibiotics in suitable concentrations can be prepared; theminimum culture times for the Govorun method are given by the knowngrowth rates.

In view of the foregoing, there is a need to provide amass-spectrometric method with which the resistance of microbes to oneor more antibiotics can be determined with certainty, at low cost, in alargely automated manner, and, most importantly, at high speed, inparticular for fast-growing and thus especially dangerous pathogens inabout an hour. It is preferable that the method can be carried out usingthe same routine mass spectrometers which are also used for themass-spectrometric identification of the microbes.

DISCLOSURE OF INVENTION

The invention provides a method for the mass-spectrometric determinationof microbial resistances, in which the microbes are cultured in a mediumcomprising an antibiotic, and a mass spectrum MS_(cum) of the microbesis acquired after they have been cultured. The method according to theinvention is characterized by the fact that any microbial growth takingplace during the culture is determined with the aid of a referencesubstance, which is added in dosed amount and also measured(co-measured) in the mass spectrum MS_(cum) wherein microbial growthindicates resistance to the antibiotic. Microbial growth can beascertained by determining the quantity of microbes after culturingusing mass spectrometry and then comparing this quantity with acorrespondingly determined quantity of microbes before culturing and/orwith a correspondingly determined quantity of microbes after culturingwithout the antibiotic.

A first preferred embodiment comprises determining the quantity ofmicrobes after the culturing from the mass spectrum MS_(cum) andreporting the microbes as being resistant if the quantity of microbesexceeds a specified limit value. It is advantageous here for thequantity of microbes to be standardized at the start of the culture.

A second preferred embodiment comprises additionally culturing themicrobes in the medium without the antibiotic and acquiring a massspectrum MS_(sine) of the microbes after their culturing without theantibiotic. The reference substance, of which a dosed amount was added,is also measured in the mass spectrum, and the microbes are reported asresistant if the quantities of microbes derived from the mass spectraMS_(cum) and MS_(sine) differ in relative or absolute terms by less thana specified limit value.

A third preferred embodiment comprises additionally acquiring a massspectrum MS₀ before culturing. The reference substance, of which a dosedamount was added, is also measured in the mass spectrum, and themicrobes are reported as being resistant if the quantity of microbesdetermined from the mass spectrum MS_(cum) significantly exceeds thequantity of microbes determined from the mass spectrum MS₀. In thisembodiment the microbes can be added to the medium, which is afterwardsdivided into two (preferably equal-sized) cultures, which are used forculturing with the antibiotic and for the acquisition of the massspectrum MS₀ respectively. On the other hand, a portion of the mediumcan also be removed after the antibiotic has been added, or at the sametime, and used for acquiring the mass spectrum MS₀.

The decision criteria used in the three preferred embodiments can, ifapplicable, also be combined by logical operators, such as an ANDoperation or a non-exclusive OR operation.

The term “mass spectrum” comprises the mass-spectrometric raw data rightthrough to a processed peak list which comprises only the positions andintensities of mass signals. A mass spectrum here can consist of a largenumber of intensity values in a continuous mass range, but also theintensity values of several separate mass ranges. The mass spectrum canbe subjected to signal processing before the quantity of microbes isdetermined. This processing can, for example, comprise correction(subtraction) of the base line, smoothing of mass signals, eliminationof noise signals and/or selection of mass signals above a specifiednoise value.

The quantity of microbes, or a measure of it, can be determined from amass spectrum in different ways, for example by the quotient of theintensity of a microbe signal and the intensity of a reference signal orthe summed intensities of several reference signals; by the quotient ofthe summed intensities of several microbe signals and the intensity of areference signal or the summed intensities of several reference signals;or by the quotient of the summed intensities of all signals of a part orthe whole mass spectrum and the intensity of a reference signal or thesummed intensities of several reference signals. Before the summation,microbe signals can be selected from a peak list or a continuous massspectrum, for example those which occur frequently in repeatmeasurements or those with a high signal intensity. In addition to peaklists, it is also possible to use mass-spectrometric raw data (evenwithout a calibrated mass axis if the positions of the referencesubstances are known) to determine the quantity of microbes, for exampleby summing all intensity values in a selected range of the raw data anddividing by the summed intensity values in a range with a referencesignal, with the base line being corrected in advance, where necessary.

The microbes can be cultured in a medium to which a single antibiotic ora mixture of different antibiotics has been added. In order todetermine, or at least estimate, the strength of the resistance, themicrobes can be cultured in parallel in several (two or more) cultures,each with a different concentration of an antibiotic, where a massspectrum MS_(cum,i) of the microbes and the dosed reference substancesis acquired for each culture. To test resistance to several antibiotics,it is possible to simultaneously (in parallel) prepare several (two ormore) cultures with several antibiotics, where necessary even withdifferent concentration levels of the antibiotics in each case. Hereagain, a mass spectrum MS_(cum,i) of the microbes and the dosedreference substances is acquired for each culture.

The reference substance can be added in a dosed amount to the mediumbefore, during or after culturing, during the preparation of amass-spectrometric sample or during the acquisition of the massspectrum. The dosed reference substance can be added after the celllysis of the microbes. If the reference substance is added to themedium, it must not be taken up or broken down by the microbes; if it isadded after the cell lysis, this condition does essentially not apply.If the mass spectra are acquired using ionization by matrix-assistedlaser desorption (MALDI), the reference substance is preferably addedonly when the MALDI samples are being prepared on a sample support, inparticular together with the matrix solution.

It is also possible to add a mixture of reference substances in dosedamounts. It is advantageous here if a larger range of concentrations iscovered by the reference substances in the mixture; three referencesubstances can be used in ratios of 100:10:1 or 25:5:1, for example. Areference substance should be able to be ionized efficiently and provideseveral identifiable reference signals in the corresponding massspectra, if possible. It is preferable if the proton affinity of thereference substance is greater than the proton affinity of most of themicrobe proteins. Preferred reference substances are ribonucleases orlysozymes. The mass of the reference substances is preferably between 10and 20 kilodaltons, in particular between 14 and 15 kilodaltons.

The microbes under analysis are preferably present in a sufficientlypure form. They can form colonies on an agar, for example, or can beobtained from a blood culture. It is also possible to examine microbesof a sample under investigation, e.g. a swab of the nasal mucosa,directly or after a pre-multiplication in a liquid medium, using themethod according to the invention. In order to carry out the methodaccording to the invention, it is not in principle necessary that thequantity of microbes is visible to the naked eye before or after theculture, i.e. before culturing, possibly less than 10⁵ microbial cells,in particular less than 10⁴ microbial cells, are sufficient, thecultures preferably being carried out in volumes between 1 μl and 1 ml,in particular in 100 μl. A low number of microbial cells in a sampleunder investigation at the beginning and a short culture time can makeit necessary to concentrate the proteins of the microbes before orduring the preparation of a mass-spectrometric sample. The concentrationcan be done, for example, by precipitating the proteins after lysing themicrobial cells with subsequent separation of the proteins, e.g. bycentrifuging, or with the aid of surfaces functionalized for proteinbinding, as can be present on (magnetic) beads, on MALDI samplesupports, or on the surface of the packing material of pipette tips, forexample. Beads separated and loaded with proteins can be applieddirectly onto a MALDI sample support, where necessary.

The method according to the invention can be used to determine theresistance of bacteria, and also to determine the resistance ofunicellular fungi, such as yeasts, to an antimycotic or a mixture ofantimycotics. A further embodiment of the method according to theinvention comprises taxonomically identifying the microbes beforedetermining the resistance and selecting the reference substance, limitvalues for the determination of the resistance, the medium and/or theculture conditions, in particular the culture period, on the basis ofthe taxonomic classification.

One embodiment of the invention is similar to the method according toGovorun, but is directed to be able to measure the microbial growth byincreases in proteins, in particular. In order to quantitativelydetermine the microbial growth in the media with antibiotics with theaid of their mass spectra, one or more suitable, precisely dosedreference substances are added to the culture or to the lysed cells. Theincrease in the biomass, and thus the proteins in particular, in mediawith antibiotics is determined with the aid of the reference substances.A comparison can be made here with the microbial growths in mediawithout antibiotics, as well as a comparison with the quantity ofmicrobes without further culturing. An increase in the biomass to anexpected extent shows that the microbes investigated are resistant tothe antibiotics at the concentration used; susceptible microbes show nomeasurable growth if the concentration of the antibiotic is above theminimum inhibitory concentration (MIC).

The method has proved to be very rapid. Since only two generations ofdoubling are necessary in order to make the biomass grow by a factor offour, the resistance of dangerous infections, which are usuallyattributable to fast-growing microbes with 20 minutes doubling time, canalready be detected after a culture period of 40 minutes; slower-growingmicrobes require longer times. The requisite culture period can bespecified when the microbe species and thus its doubling time, is known.

The invention also provides a sample support for ionization bymatrix-assisted laser desorption (MALDI) with a large number of sampleareas, on each of which is a thin layer of a MALDI matrix substance. Thesample support is characterized by the fact that the thin layercomprises a dosed reference substance with a mass of between 10 and 20kilodaltons, in particular between 14 and 15 kilodaltons. The thin layercan furthermore have at least two different reference substances, whichdiffer in concentration by a factor of between 5 and 100, if requiredalso by a factor of 1000. Furthermore, the invention provides consumablematerial for ionization by matrix-assisted laser desorption (MALDI).This consumable material is a freeze-dried mixture of a MALDI matrixsubstance and at least one reference substance. The mass of the one ormore reference substances is preferably between 10 and 20 kilodaltons,in particular between 14 and 15 kilodaltons. It is preferable if thereference substances are present in different concentrations, forexample in the ratio 5:1, 10:1, 100:1 or 1000:1.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an example of a flow diagram for a preferred method foridentifying a microbe and determining its resistance according to thisinvention.

FIG. 2 shows four mass spectra of two strains of one species of bacteriaof the genus klebsiella. The top two mass spectra originate from strain1, which is susceptible, so no growth has occurred in the culture withthe added antibiotic (“Ab”). In the second spectrum from the top(“strain 1+Ab”), practically only the mass peaks of the referencesubstance (ribonuclease A) are visible, singly charged on the far right,doubly charged slightly left of center. The bottom two mass spectra wereobtained from strain 5, which is resistant, so the growth is uninhibitedeven when the antibiotic (“strain 5+Ab”) is added (spectrum at the verybottom). All the mass spectra were acquired after a culture period ofonly one hour.

FIG. 3 illustrates an evaluation of the mass spectra of five klebsiellastrains, in each case with and without antibiotic. Strain 1 and strain 2are susceptible, therefore no growth in the presence of the antibiotic.Strains 3 and 5 are resistant, the same growth with and withoutantibiotic. For strain 4 it can be assumed either that an intermediateresistance is present or that only some of the harvested colonies areresistant, so the initial quantity of resistant microbes was smaller.The boxes represent the average variances for repeated measurements.

PREFERRED MODE(S) FOR CARRYING OUT THE INVENTION

While the invention has been shown and described with reference to anumber of embodiments thereof, it will be recognized by those skilled inthe art that various changes in form and detail may be made hereinwithout departing from the spirit and scope of the invention as definedby the appended claims.

The invention proposes a preferred method for the mass-spectrometricdetermination of the resistance of microbes by comparing the massspectra of the microbes after culturing in several media of the sametype, with and without the addition of antibiotics, wherein themicrobial growth during culture is determined with the aid of at leastone added reference substance, and microbial growth in a mediumcomprising antibiotic indicates resistance to this antibiotic at thegiven concentration. The microbes can in particular also be cultured inparallel in several (two or more) media with different types ofantibiotic.

The reference substance can already be added at a dosed amount to theculture medium, which must then be of such a kind that it is alwaystaken up by the microbes in the same quantity and is not decomposed, inorder to be visible in the preparation after rinsing and cell lysis.Since this condition is difficult to fulfill, the reference substance ispreferably added after culturing; for example it is added to the lysedcells or, in the case of ionization by matrix-assisted laser desorption,to the preparation of the sample on the sample support.

In order to determine the strength of the resistance or to determinewhether or not the microbes of all colonies harvested are resistant inthe same way, the microbes can be cultured in media comprisingantibiotics at several concentration levels. If only some of thecolonies harvested are resistant, for which the term “mixed resistance”is used here, then all levels of concentration exhibit the sameproportionate reduction in growth compared to the culture withoutantibiotic, because the growth was started with a lower quantity ofresistant microbes. This case is relatively rare, however. In the caseof intermediate resistance, on the other hand, there is no longer anygrowth if the concentration of the antibiotic is high, but full growthif the concentration is low. It is also quite possible to not mix themicrobes of the colonies, but to carry out these tests separately forthe microbes of each colony

Current practice is to cultivate microbes on agar or in a blood culturefor identification, usually overnight. With agar cultures there are thenusually several colonies, one of which is harvested for theidentification. To determine the resistance, it is common practice tosubject the microbes from at least five further colonies together tothis test in order to also detect the presence of a resistant microbecolony among non-resistant microbe colonies of the same species (mixedresistance). A trained and experienced laboratory technician isgenerally able to recognize colonies of the same microbes and to harvestthem. The microbes of these colonies can then preferably be mixed anddivided up between the different cultures. But in special cases they canalso be subjected to the resistance determination individually, forexample if it cannot be assumed with certainty that the colonies belongto the same microbial species. Microbes from blood cultures naturallycontain a mixture of different microbes which were transmitted in theinfection. As those skilled in the art know, the blood culture usuallyends in a centrifuge pellet which contains sufficient microbes for theidentification and also for the resistance determination.

In one embodiment, the invention is similar to the method by Govorun,where mass spectra of the microbes from cultures with and without addedantibiotics are compared with each other, but the invention is directedtoward measurement of the microbial growth, which Govorun does notmention. In order to quantitatively determine the microbial growth inmedia with antibiotics with the aid of their mass spectra, one or moresuitable, precisely dosed reference substances are added, preferablyafter the cell lysis. The increase in the biomass, and thus theassociated increase in the proteins in particular, in the media withantibiotics is quantitatively determined with the aid of the referencesubstances. In particular, a comparison can be made here with themicrobial growths in media without antibiotics, and also a comparisonwith the quantity of the microbes used without further culturing. Anincrease in the biomass to an expected extent shows that the microbesinvestigated are resistant to the antibiotics at the concentration used;susceptible microbes exhibit no growth if the concentration of theantibiotic is above the minimum inhibitory concentration (MIC).

Time-of-flight mass spectrometers with ionization by matrix-assistedlaser desorption (MALDI) are mainly used for the identification. Fordecades, MALDI was deemed unsuitable for quantitative analyses. But ithas long been shown that this is incorrect, and was attributable to thevery coarse preparation methods used in the past for the samples on thesample support. It can be shown that the quantity of a substance can bedetermined with MALDI with an accuracy of two percent for a very goodand uniform thin layer preparation and if reference substances are addedin suitably precise doses, for example. Nothing like this degree ofaccuracy is required here; the effort which would have to be expendedwould also complicate the routine method unnecessarily. A quantitativeaccuracy of around 20 percent can also be achieved in a routinelaboratory without much effort. Since the microbial growths in everydoubling time involve a factor of two, i.e. for two doubling times afactor of four, MALDI is easily capable of fulfilling this task ofmeasuring the microbial growths, even if the growth were to be slower inthe presence of the antibiotic, or if not all the microbe coloniesharvested are resistant.

The invention therefore proposes adding one or more substances which aresuitable as quantitative references in suitable, accurately knownquantities (or concentrations), for which the term “dosed” is used here,before measuring the mass spectra of the microbes. The referencesubstances can already be added to the culture medium; however, thisthen requires that the reference substance is preferably always taken upby the microbes in the same quantity and is not destroyed by digestion.It is therefore advantageous to add the dosed reference substances onlyafter the microbes have been killed, either to the proteins in theliquid of the disrupted microbes in the centrifuge tube or to the matrixsolution which is applied to the dried proteins on the MALDI samplesupport. These reference substances make it possible to quantitativelyestimate the relative growth of the microbes with and without theaddition of antibiotics, and to determine the resistance orsusceptibility from this. The accuracy for the quantities andconcentrations used should preferably be around 10 percent in order tomaintain the overall accuracy of the method at around 20 percent. Thereference substances should be easily ionizable due to a high protonaffinity so that their ionization cannot be suppressed by the proteinsof the microbes. Where possible, they should provide several easilyrecognizable peaks in the mass spectra. It is advantageous if thereference substances cover a larger range of quantities; three referencesubstances can be used in ratios of 100:10:1 or 25:5:1, for example. Ithas proved to be advantageous if the reference substance with thehighest concentration produces reference signals in the mass spectrumwhich are around the same height (intensity) as exhibited by the highestmicrobe signals (usually protein signals) after full, uninhibitedgrowth.

Ribonuclease A is cited here as an example of a substance which can beused successfully. It has a molecular weight of 13,638 daltons; its highproton affinity has the effect that singly, and also doubly and eventriply, charged ions of ribonuclease A appear in the MALDI spectrum. Thesingly charged RNase-A ions appear in an area of the mass spectrum wherethey are easily recognizable, usually without interferences from otherpeaks.

Other ribonucleases can be given here as further suitable substances,for example. But it is also possible to use other substance classes withhigh proton affinity, for example lysozymes. Lysozyme C has a molecularmass of 14.3 kilodaltons, and so also has a peak in a sparsely populatedrange of the mass spectrum.

The method has proved to be surprisingly rapid: dangerous infections areusually caused by rapidly growing microbes with a doubling time of onlyabout 20 minutes. Since only two generations of doubling are necessaryto make the biomass grow by a factor of four, the resistance of thesefast-growing microbes can already be identified after a culture periodof only around 40 minutes; slower-growing microbes with 30 minutesdoubling time require an hour. If one adds another 20 minutes forprocessing the microbes, preparation for MALDI ionization and acquiringthe mass spectra, the resistance can be known in between one and one anda half hours after their identification. It is advantageous here thatthe microbes were identified before the determining the resistance. Whenthe microbe species and its doubling time are known, the requiredculture period can be optimally specified.

The method further provides possibilities to check against mistakes madewhen the microbes are harvested or the samples prepared. The massspectra of the microbes acquired after they have been cultured can besubjected once again to the identification routine for the microbes inorder to confirm the correct assignment. Since this method ofidentification is time-consuming, it can be accelerated by simplydetermining the similarities between the mass spectra acquired afterculturing and the mass spectrum which was used for the identification.The similarity values provide information on whether the correct massspectra are present for determining the resistance.

Between full resistance of the microbes and full susceptibility thereare intermediate stages; the growth is impaired, but not completelyinhibited. In order to determine, or at least estimate, the strength ofthe resistance of microbes, the actual inhibitory concentrations of theantibiotics can be measured. The MIC values of the antibiotics (minimuminhibitory concentrations for fully susceptible microbes) are known to alarge extent; the actual inhibitory concentrations increase with thestrength of the resistance, however. To measure the actual inhibitoryconcentrations, cultures can be used to which an antibiotic at variousconcentration levels is added, wherein the concentration levels cancorrespond to the concentration 1*MIC, 10*MIC and 100*MIC of the knownMIC values, for example. In our experience, the inhibition of microbialgrowth at a concentration of 1*MIC can only be detected with the methoddescribed above if the microbes are fully susceptible. In case of a weakresistance, the microbes are inhibited only at a concentration of10*MIC, while for a very strong resistance, growth can still be detectedeven at a concentration of 100*MIC. The effect can be seen from thevalues of the microbial growths. With intermediate resistances there istherefore different growth at different concentrations of theantibiotic.

It can also be the case, however, that only a proportion, for examplehalf, of the colonies harvested are resistant and the others aresusceptible. We call this a “mixed resistance”. Less growth then seemsto be detected even with strong resistance, but only because there werea smaller number of resistant microbes in the culture at the start. Ifthe test here is carried out with different concentrations of theantibiotic, all concentrations exhibit the same percentage reduction inprotein growth compared to the culture without antibiotic

If the method is carried out without concentration levels, aconcentration of 10*MIC has proved to be particularly suitable.

To test the resistance to several antibiotics, it is possible to prepareseveral cultures with several antibiotics, where necessary even withdifferent concentration levels of the antibiotics in each case. Theadditional time needed to prepare the microbes from several (two ormore) cultures is of no consequence compared to the time required forthe culture.

For a rapid test for multi-resistant germs (example: MRSA,methicillin-resistent staphylococcus aureus), a mixture of several typesof antibiotics can be added to the media. If the microbes grow in thismixture, they are multiresistant. In this rapid test, a sample underinvestigation, such as a swab of the nasal mucosa, can also comprise amixture of microbes and there is no need to identify the microbes in thesample beforehand. The mass spectrum acquired can be used to identifythe microbes grown in the medium comprising antibiotic and thusdetermined as being resistant.

In a preferred embodiment, reactive substances are additionally added tothe culture medium with the antibiotic in order to get a better accuracyof discrimination between resistant and susceptible microbes. Thereactive substances can reactively modify microbes which are alreadyweakened by the antibiotic and thus boost and assist the effect of theantibiotic. It is possible, for example, to add enzymes which can attackand destroy microbes whose growth has been affected while unaffectedmicrobes cannot be attacked by the enzymes.

In a preferred method, the intensities of all the mass peaks in aselected section of the spectrum, for example from 4,000 to 10,000daltons, are summed and divided by the intensity of the peak of thesingly charged reference ions: this gives the quotient q. It is possibleto refine the routine method for resistance determination in such a waythat values of this quotient q under 200 represent susceptible peakswithout growth (many of the peaks here are noise); while values of thequotient q above 200 indicate resistance. More preferable is anevaluation which forms the quotients Q=q_(cum)/q_(sine) for microbes inmedia with antibiotics (q_(cum)) and without antibiotics (q_(sine)). Ifthis quotient is close to one (0.8<Q<1.3), the microbes are resistant; Qis approximately 0.25 (0.1<Q<0.4) for susceptible germs if the cultureperiod is around two doubling times.

In another preferred method, the mass spectrum MS_(cum) acquired frommicrobes after they have been cultured in a medium with antibiotic isnormalized to the maximum signal, if necessary after a base linesubtraction. Multiple thresholds between zero and one are selected,preferably more than 10 and more preferably about 100 equidistantthresholds. For each threshold, peaks with intensity above the thresholdare determined and the number of the determined peaks is assigned to thecorresponding threshold resulting in a curve (numbers of peaks abovethreshold vs. threshold). The step of determining the number of peaksabove threshold is preferably performed on a peak list obtained from thenormalized and base line subtracted mass spectrum MS_(cum). The areaunder the normalized curve (AUC) or the minimum distance between thepoint of origin and the curve can be determined as preferable measuresfor the microbial growth. An AUC above a specified value may indicatethe resistance of the microbes against the antibiotic. More preferably,the AUC is also determined for MS_(sine), i.e. the same signalprocessing is also applied to a mass spectrum MS_(sine) of microbeswhich are cultivated under the same conditions, but without anyantibiotic being present. A ratio AUC(MS_(cum))/AUC(MS_(sine)) above aspecific value (preferably 4/10) indicates resistance against theantibiotic under investigation.

For routine laboratories with a larger number of microbe samples whichmust be both identified and tested for their resistance, it is valuableto be able to automate at least parts of the procedural steps. Acomplete automation of the whole method is currently not yet possible;there are, however, a number of instruments already on the market orunder development and close to market readiness which can handle atleast some procedural steps automatically or semi-automatically. Thereis an instrument which can harvest microbe colonies from agar plateseither under visual control or by image analysis and which can apply themicrobes onto the sample support plate for identification. Thisinstrument can easily be developed further in order to also harvestmicrobes for determining the resistance. Instruments for lysing ofmicrobes on the sample support plate and the preparation with matrixsolution are also conceivable. Pipetting robots are available which cancarry out the lysis of centrifuge pellets in suitable microtitrationplates or in series of centrifuge tubes. Culturing can be carried out incentrifuge tubes (for example Eppendorf tubes) or in filter plates (forexample Acropep 96-well filter plates). IVD-certified methods for MALDImass spectrometers which operate with sample supports being able to hold48, 96 or 384 samples are commercially available.

Ionization by matrix-assisted laser desorption (MALDI) requires either asample support plate on which the matrix substance is already preparedin a thin layer, or preparation of a matrix solution. Commercially soldmatrix substances often have the disadvantage that they are difficult todissolve without ultrasound. Therefore, small bottles with purified andfreeze-dried matrix substances in accurately dosed quantities areavailable, in which the matrix substance dissolves immediately when thesolvent is added; the solution is ready to use immediately in thecorrect concentration. According to this invention, at least onereference substance for the quantification of protein increase can beadded to the matrix substances of these products in the correct dose. Inthe device for the preparation of MALDI samples, the matrix solution canbe applied to the dried cell components of the microbes, especiallyproteins, in a proper dose and without coming into contact with them.The sample support plates with thin matrix layers which are already soldcommercially can also comprise reference substances in dosed amounts.The thin layers are each applied to small sample areas which are wellseparated from each other and each have a diameter of around twomillimeters.

The sequence of a preferred method for determining resistances is shownin the diagram of FIG. 1 as an example. The method is shown here withthe microbes being cultured on an agar (101). The microbes of a colonyare harvested (102), disrupted (lysed), and processed into a MALDIsample (103). The acquisition of a mass spectrum (104) leads to theidentification of the microbe by comparing its mass spectrum withreference spectra (105). In a routine laboratory it takes only 10 to 30minutes from the harvesting of a colony through to the identification,depending on the number of microbe samples to be identified in parallel.It is possible to harvest several further colonies of the same microbeat the same time (106) in order to determine the resistance. Thesecolonies are mixed and divided up between the different types of culture(107). Three cultures are prepared in the example shown in this diagram:one culture in a medium without antibiotic (108), and two cultures withthe antibiotics Ab1 (109) and Ab2 (110). Of course, further cultureswith further antibiotics and, if the strength of the resistance is alsoto be determined, cultures with different concentrations of theantibiotics can be prepared. All cultures are already prepared at theoptimum temperature in order not to subject the microbes to a shock andso that the heating does not cause a time delay. The duration of theculture depends on the doubling time (generation time) of the microbes,which is known from the identification of the microbes. The culture onlyneeds to last two to three doubling times. Around 40 minutes aresufficient for fast-growing microbes. The microbes from the differentcultures are processed into MALDI samples with the addition of referencesubstances, and mass spectra are acquired (111). The quotient q_(sine)is formed from the mass spectrum of the microbes which were culturedwithout antibiotics, by adding the intensities of all peaks in the massrange of 4,000 to 10,000 daltons, for example, and then dividing by theintensity of the reference peak. The corresponding quotients q_(cum,n)are formed (112) from the mass spectra of the microbes which werecultured with the respective antibiotic n. As was stated above, thequotients Q_(n)=q_(cum,n)/q_(sine) show the resistance in each case.

The methods have to date been carried out with ionization by MALDI in aMALDI time-of-flight mass spectrometer. MALDI has the great advantagethat it forms almost only singly charged molecular ions. Therefore themass spectra are not overloaded despite the 50 to 100 peaks which appearin the preferred mass range from 3,000 to 15,000 daltons, andsimilarities can be recognized relatively easily. This does not mean,however, that it is not possible to use other types of ionization. Thespray-based methods, such as ESI (electrospray ionization) or DESI(direct surface ionization of solid samples by electrospray, desorptionelectrospray ionization), form multiply charged ions, which can easilyoverload the mass spectra, but they can be coupled with separationmethods such as liquid chromatography (HPLC) or capillaryelectrophoresis (CE) so that it is possible to again obtain mass spectrawith a simpler composition by separating the substances.

There are, however, other ionization methods which also produce almostonly singly charged ions, for example chemical ionization (CI). Chemicalionization can be used in conjunction with neutral spray methods, butalso with laser ablation of solid samples, and can be employed inconjunction with an OTOF-MS (time-of-flight mass spectrometer withorthogonal ion injection). The mass spectra thus obtained provideextremely high mass resolution with high sensitivity (cf. J. Franzen andK. Michelmann, DE 10 2005 044 307 B4, for example).

It is, of course, also possible to use other types of mass spectrometerif they provide the preferred mass range of 3,000 to 15,000 daltons formeasuring the mass spectra.

Different aspects of the invention have been elucidated above. It willbe understood, however, that various aspects or details of the inventionmay be changed, or that different aspects disclosed in conjunction withdifferent embodiments of the invention may be readily combined ifpracticable, without departing from the scope of the invention.Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limiting the inventionwhich is defined solely by the appended claims.

The invention claimed is:
 1. A method for the mass-spectrometricdetermination of the resistance of microbes to an antibiotic, in whichthe microbes are cultured in a medium comprising the antibiotic, and amass spectrum MS_(cum) of the microbes is acquired after they have beencultured, the method comprising: providing at least one referencesubstance in a dosed amount that is measured together with the culturedmicrobes such that the mass spectrum MS_(cum) comprises one or morereference mass signals of the at least one reference substance; anddetermining microbial growth taking place during the culture from theintensity of one or more microbe signals of the mass spectrum MS_(cum),using the intensity of one or more of the reference mass signals as aquantitative reference, said microbial growth indicating the resistanceto the antibiotic, wherein the mass spectrum MS_(cum) is of the range of2,000 to 15,000 daltons.
 2. The method according to claim 1, wherein aquantity of microbes is standardized at the start of the culturing, andis subsequently determined after culturing from an intensity of one ormore microbe signals of the mass spectrum MS_(cum) using the intensityof one or more of the reference mass signals, and wherein the microbesare reported as being resistant to the antibiotic if the quantity ofmicrobes exceeds a specified limit value.
 3. The method according toclaim 1, further comprising: preparing a second culture of the microbesin the medium without the antibiotic; acquiring a mass spectrumMS_(sine) of the microbes of the second culture wherein the at least onereference substance is added in a dosed amount to the medium withoutantibiotic before, during or after culturing, or is added in a dosedamount during the preparation of the mass-spectrometric sample or duringacquisition of the mass spectrum such that it is measured in the massspectrum MS_(sine); and determining the microbes as being resistant tothe antibiotic if the quantities of microbes derived from the intensityof one or more microbe signals of the mass spectra MS_(cum) andMS_(sine) using the intensity of one or more reference signals of themass spectra MS_(cum) and MS_(sine) as a quantitative reference differin relative or absolute terms by less than a specified limit value. 4.The method according to claim 3, wherein the quantity of microbes isdetermined as a quotient from a mass spectrum, wherein the quotient isformed from one of the intensity of a microbe signal and the intensityof a reference signal, the intensity of a microbe signal and the summedintensities of several reference signals, the summed intensities ofseveral microbe signals and the intensity of a reference signal, thesummed intensities of several microbe signals and the summed intensitiesof several reference signals, the summed intensities of all the signalswithin one region or within the whole mass spectrum and the intensity ofa reference signal, and the summed intensities of all the signals withinone region or within the whole mass spectrum and the summed intensitiesof several reference signals.
 5. The method according to claim 1,further comprising: acquiring an additional mass spectrum MS₀ of themicrobes before the microbes are cultured; adding the at least onereference substance in a dosed amount to the medium or during thepreparation of the mass-spectrometric sample or during acquisition ofthe mass spectrum such that it is measured in the mass spectrum MS₀; anddetermining the microbes as being resistant to the antibiotic if thequantity of microbes determined from the intensity of one or moremicrobe signals of the mass spectrum MS_(cum) significantly exceeds thequantity of microbes being determined from the intensity of one or moremicrobe signals of the mass spectrum MS₀ using the intensity of one ormore reference signals of the mass spectrum MS₀ as a quantitativereference.
 6. The method according to claim 1, wherein the mass spectraare acquired by means of matrix-assisted laser desorption/ionization(MALDI), and the at least one reference substance is added duringpreparation of the MALDI samples on a sample support.
 7. The methodaccording to claim 1, wherein the microbes are cultured simultaneouslyin several cultures, each having a different antibiotic, and a massspectrum MS_(cum) is acquired for each culture.
 8. The method accordingto claim 1, wherein the microbes are simultaneously cultured in severalcultures, each having a different concentration of the antibiotic, and amass spectrum MS_(cum) is acquired for each culture.
 9. The methodaccording to claim 1, wherein the proton affinity of the at least onereference substance is greater than the proton affinity of most of theproteins of the microbes.
 10. The method according to claim 9, whereinthe at least one reference substance is a ribonuclease or a lysozyme.11. The method according to claim 1, wherein the proteins of themicrobes are concentrated before or during the preparation of amass-spectrometric sample.
 12. The method according to claim 1, whereina mixture of reference substances is added in a dosed amount to themedium before, during or after culturing, or is added in a dosed amountduring the preparation of the mass-spectrometric sample or duringacquisition of the mass spectrum such that they are measured in the massspectrum MS_(cum).
 13. The method according to claim 12, wherein thereference substances of the mixture are present in differentconcentrations which differ by a factor between 5 and
 100. 14. Themethod according to claim 1, wherein the microbes are identifiedtaxonomically before their resistance is determined, and the at leastone reference substance, limit values for determining the resistance,the medium and the culture conditions are selected on the basis of thetaxonomic classification.
 15. The method according to claim 1, whereinthe mass spectra are acquired by means of matrix-assisted laserdesorption/ionization (MALDI).
 16. The method according to claim 15,wherein multiple MALDI samples are prepared on a sample support on whicha matrix substance is already prepared in a layer wherein the layer hasa dosed addition of a reference with a mass of between 10 and 20kilodaltons.
 17. The method according to claim 1, wherein the mass ofthe at least one reference substance is between 10 and 20 kilodaltons.18. A method for the mass-spectrometric determination of the resistanceof microbes to an antibiotic, in which the microbes are cultured in amedium comprising the antibiotic, and a mass spectrum of the microbes isacquired after they have been cultured, wherein: at least one referencesubstance is added in a dosed amount to the medium before, during orafter culturing, or is added in a dosed amount during the preparation ofthe mass-spectrometric sample or during acquisition of the mass spectrumsuch that it is measured as at least one reference mass signal in themass spectrum; microbial growth is ascertained by determining thequantity of microbes after culturing from the mass spectrum and thencomparing this quantity with a correspondingly determined orstandardized quantity of microbes before culturing and/or with acorrespondingly determined quantity of microbes after culturing withoutthe antibiotic; and microbial growth indicates the resistance to theantibiotic, wherein the quantity of microbes is determined from theintensity of the at least one reference mass signal of the at least onereference substance, and the mass spectrum is of the range of 2,000 to15,000 daltons.
 19. The method according to claim 18, wherein the massspectra are acquired by means of matrix-assisted laserdesorption/ionization (MALDI).
 20. The method according to claim 19,wherein the mass of the at least one reference substance is between 10and 20 kilodaltons.